Mutation in the Jak kinase JH2 domain hyperactivates Drosophila and mammalian Jak-Stat pathways.

The Jak (Janus) family of nonreceptor tyrosine kinases plays a critical role in cytokine signal transduction pathways. In Drosophila melanogaster, the dominant hop(Tum-l) mutation in the Hop Jak kinase causes leukemia-like and other developmental defects. Previous studies have suggested that the Hop(Tum-l) protein might be a hyperactive kinase. Here, we report on the new dominant mutation hop(T42), which causes abnormalities that are similar to but more extreme than those caused by hop(Tum-l). We determined that Hop(T42) contains a glutamic acid-to-lysine substitution at amino acid residue 695 (E695K). This residue occurs in the JH2 (kinase-like) domain and is conserved among all Jak family members. We determined that Hop(Tum-1) and Hop(T42) both hyperphosphorylated and hyperactivated D-Stat when overexpressed in Drosophila cells. Moreover, we found that the hop(T42) phenotype was partially rescued by a reduction of wild-type D-stat activity. Finally, generation of the corresponding E695K mutation in murine Jak2 resulted in increased autophosphorylation and increased activation of Stat5 in COS cells. These results demonstrate that the mutant Hop proteins do indeed have increased tyrosine kinase activity, that the mutations hyperactivate the Hop-D-Stat pathway, and that Drosophila is a relevant system for the functional dissection of mammalian Jak-Stat pathways. Finally, we propose a model for the role of the Hop-D-Stat pathway in Drosophila hematopoiesis.

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The Thrombopoietin Receptor Can Mediate Proliferation without Activation of the Jak-STAT Pathway

Journal of Experimental Medicine ◽

10.1084/jem.186.12.1947 ◽

1997 ◽

Vol 186 (12) ◽

pp. 1947-1955 ◽

Cited By ~ 45

Author(s):

Marion Dorsch ◽

Pang-Dian Fan ◽

Nika N. Danial ◽

Paul B. Rothman ◽

Stephen P. Goff

Keyword(s):

Tyrosine Kinases ◽

Kinase Inhibitor ◽

Mitogen Activated Protein Kinase ◽

Signal Transducers ◽

Thrombopoietin Receptor ◽

Mitogen Activated Protein ◽

Mutant Receptors ◽

Nonreceptor Tyrosine Kinases ◽

Stat Pathway

Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.

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Calcium Signaling in Vertebrate Development and Its Role in Disease

International Journal of Molecular Sciences ◽

10.3390/ijms19113390 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3390 ◽

Cited By ~ 8

Author(s):

Sudip Paudel ◽

Regan Sindelar ◽

Margaret Saha

Keyword(s):

Calcium Signaling ◽

Critical Role ◽

Molecular Techniques ◽

Model Organisms ◽

Vertebrate Development ◽

Developmental Defects ◽

Calcium Activity ◽

Human Genes ◽

Underlying Mechanisms

Accumulating evidence over the past three decades suggests that altered calcium signaling during development may be a major driving force for adult pathophysiological events. Well over a hundred human genes encode proteins that are specifically dedicated to calcium homeostasis and calcium signaling, and the majority of these are expressed during embryonic development. Recent advances in molecular techniques have identified impaired calcium signaling during development due to either mutations or dysregulation of these proteins. This impaired signaling has been implicated in various human diseases ranging from cardiac malformations to epilepsy. Although the molecular basis of these and other diseases have been well studied in adult systems, the potential developmental origins of such diseases are less well characterized. In this review, we will discuss the recent evidence that examines different patterns of calcium activity during early development, as well as potential medical conditions associated with its dysregulation. Studies performed using various model organisms, including zebrafish, Xenopus, and mouse, have underscored the critical role of calcium activity in infertility, abortive pregnancy, developmental defects, and a range of diseases which manifest later in life. Understanding the underlying mechanisms by which calcium regulates these diverse developmental processes remains a challenge; however, this knowledge will potentially enable calcium signaling to be used as a therapeutic target in regenerative and personalized medicine.

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Snapin deficiency is associated with developmental defects of the central nervous system

Bioscience Reports ◽

10.1042/bsr20100110 ◽

2010 ◽

Vol 31 (2) ◽

pp. 151-158 ◽

Cited By ~ 8

Author(s):

Bing Zhou ◽

Yi-Bing Zhu ◽

Lin Lin ◽

Qian Cai ◽

Zu-Hang Sheng

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Protein Quality ◽

Third Ventricle ◽

Critical Role ◽

Developmental Defects ◽

Lysosomal Function ◽

Quality Control Process ◽

The Central Nervous System

The autophagy–lysosomal pathway is an intracellular degradation process essential for maintaining neuronal homoeostasis. Defects in this pathway have been directly linked to a growing number of neurodegenerative disorders. We recently revealed that Snapin plays a critical role in co-ordinating dynein-driven retrograde transport and late endosomal–lysosomal trafficking, thus maintaining efficient autophagy–lysosomal function. Deleting snapin in neurons impairs lysosomal proteolysis and reduces the clearance of autolysosomes. The role of the autophagy–lysosomal system in neuronal development is, however, largely uncharacterized. Here, we report that snapin deficiency leads to developmental defects in the central nervous system. Embryonic snapin−/− mouse brain showed reduced cortical plates and intermediate zone cell density, increased apoptotic death in the cortex and third ventricle, enhanced membrane-bound LC3-II staining associated with autophagic vacuoles and an accumulation of polyubiquitinated proteins in the cortex and hippocampus. Thus our results provide in vivo evidence for the essential role of late endocytic transport and autophagy–lysosomal function in maintaining neuronal survival and development of the mammalian central nervous system. In addition, our study supports the existence of a functional interplay between the autophagy–lysosome and ubiquitin–proteasome systems in the protein quality-control process.

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Osmotic shrinkage elicits FAK- and Src phosphorylation and Src-dependent NKCC1 activation in NIH3T3 cells

AJP Cell Physiology ◽

10.1152/ajpcell.00070.2014 ◽

2015 ◽

Vol 308 (2) ◽

pp. C101-C110 ◽

Cited By ~ 6

Author(s):

Line Jee Hartmann Rasmussen ◽

Helene Steenkær Holm Müller ◽

Bente Jørgensen ◽

Stine Falsig Pedersen ◽

Else Kay Hoffmann

Keyword(s):

Focal Adhesion ◽

Tyrosine Kinases ◽

Volume Regulation ◽

Mammalian Cells ◽

Myosin Light Chain ◽

Janus Kinase ◽

Nih3t3 Cells ◽

Src Inhibitor ◽

Nonreceptor Tyrosine Kinases

The mechanisms linking cell volume sensing to volume regulation in mammalian cells remain incompletely understood. Here, we test the hypothesis that activation of nonreceptor tyrosine kinases Src, focal adhesion kinase (FAK), and Janus kinase-2 (Jak2) occurs after osmotic shrinkage of NIH3T3 fibroblasts and contributes to volume regulation by activation of NKCC1. FAK phosphorylation at Tyr397, Tyr576/577, and Tyr861 was increased rapidly after exposure to hypertonic (575 mOsm) saline, peaking after 10 (Tyr397, Tyr576/577) and 10–30 min (Tyr861). Shrinkage-induced Src family kinase autophosphorylation (pTyr416-Src) was induced after 2–10 min, and immunoprecipitation indicated that this reflected phosphorylation of Src itself, rather than Fyn and Yes. Phosphorylated Src and FAK partly colocalized with vinculin, a focal adhesion marker, after hypertonic shrinkage. The Src inhibitor pyrazolopyrimidine-2 (PP2, 10 μM) essentially abolished shrinkage-induced FAK phosphorylation at Tyr576/577 and Tyr861, yet not at Tyr397, and inhibited shrinkage-induced NKCC1 activity by ∼50%. The FAK inhibitor PF-573,228 augmented shrinkage-induced Src phosphorylation, and inhibited shrinkage-induced NKCC1 activity by ∼15%. The apparent role of Src in NKCC1 activation did not reflect phosphorylation of myosin light chain kinase (MLC), which was unaffected by shrinkage and by PP2, but may involve Jak2, a known target of Src, which was rapidly activated by osmotic shrinkage and inhibited by PP2. Collectively, our findings suggest a major role for Src and possibly the Jak2 axis in shrinkage-activation of NKCC1 in NIH3T3 cells, whereas no evidence was found for major roles for FAK and MLC in this process.

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FGFR3 Associates with and Tyrosine-Phosphorylates p90RSK2, Leading to RSK2 Activation That Mediates Hematopoietic Transformation

Blood ◽

10.1182/blood.v112.11.3722.3722 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3722-3722

Author(s):

Sumin Kang ◽

Shannon Elf ◽

Shaozhong Dong ◽

Taro Hitosugi ◽

Ailan Guo ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Mapk Pathway ◽

Critical Role ◽

Ectopic Expression ◽

Alpha Helix ◽

Tail Region ◽

Murine Bone Marrow

Abstract Dysregulation of receptor tyrosine kinase FGFR3 has been implicated to play a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates p90 Ribosomal S6 Kinase2(RSK2) at Y529, which consequently regulates RSK2 activation [Kang et al, Cancer Cell 2007 Sep;12(3):201–14]. Here we identified Y707 as an additional tyrosine site of RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation, through a putative disruption of the autoinhibitory αL-helix on the C-terminus of RSK2, unlike Y529 phosphorylation that facilitates ERK binding. To elucidate the role of tyrosine phosphorylation at Y707 induced by FGFR3 in RSK2 activation, we characterized the RSK2 mutants with single Y→A and Y→F substitutions at Y707. RSK2 Y707F demonstrated decreased kinase activity, suggesting substitution of Y707 attenuates activation of RSK2 induced by FGFR3. Tyrosine phosphorylation at Y529 by FGFR3 regulates RSK2 activation by facilitating inactive ERK binding, whereas substitution of Y707 in RSK2 does not similarly attenuate inactive ERK binding to RSK2. Phosphorylation at Y707 may regulate RSK2 activation by affecting the structure of the autoinhibitory C-terminal domain of RSK2 since the Y707 is localized at the C-terminal tail region which represents a conserved putative auto-inhibitory alpha helix. Since other tyrosine kinases including FGFR1 and Src also phosphorylate RSK2 at Y529 and Y707, tyrosine phosphorylation may be a general requirement for RSK2 activation through the ERK/MAPK pathway. Together, our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases. Moreover, we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2, and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707, and subsequent RSK2 activation. Furthermore, in a murine bone marrow transplant assay, genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild type cells, suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation.

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Emerging roles of PLCγ1 in endothelial biology

Science Signaling ◽

10.1126/scisignal.abc6612 ◽

2021 ◽

Vol 14 (694) ◽

pp. eabc6612

Author(s):

Dongying Chen ◽

Michael Simons

Keyword(s):

Tyrosine Kinases ◽

Second Messengers ◽

Critical Role ◽

Physiological Role ◽

Membrane Lipid ◽

Molecular Function ◽

Signal Transducer ◽

Deficient Animal ◽

Unique Protein

Phospholipase C γ1 (PLCγ1) is a member of the PLC family that functions as signal transducer by hydrolyzing membrane lipid to generate second messengers. The unique protein structure of PLCγ1 confers a critical role as a direct effector of VEGFR2 and signaling mediated by other receptor tyrosine kinases. The distinct vascular phenotypes in PLCγ1-deficient animal models and the gain-of-function mutations of PLCγ1 found in human endothelial cancers point to a major physiological role of PLCγ1 in the endothelial system. In this review, we discuss aspects of physiological and molecular function centering around PLCγ1 in the context of endothelial cells and provide a perspective for future investigation.

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Regulation of Discrete Functional Responses by Syk and Src Family Tyrosine Kinases in Human Neutrophils

Journal of Immunology Research ◽

10.1155/2017/4347121 ◽

2017 ◽

Vol 2017 ◽

pp. 1-7 ◽

Cited By ~ 4

Author(s):

Thornin Ear ◽

Olga Tatsiy ◽

Frédérick L. Allard ◽

Patrick P. McDonald

Keyword(s):

Translational Control ◽

Tyrosine Kinases ◽

Critical Role ◽

Protein Tyrosine Kinases ◽

Human Neutrophils ◽

Functional Responses ◽

Adaptive Immune ◽

Inflammatory Conditions ◽

Src Family Tyrosine Kinases

Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs), as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1) involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.

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Ras and Rab interactor 1 controls neuronal plasticity by coordinating dendritic filopodial motility and AMPA receptor turnover

Molecular Biology of the Cell ◽

10.1091/mbc.e16-07-0526 ◽

2017 ◽

Vol 28 (2) ◽

pp. 285-295 ◽

Cited By ~ 11

Author(s):

Zsófia Szíber ◽

Hanna Liliom ◽

Carlos O. Oueslati Morales ◽

Attila Ignácz ◽

Anikó Erika Rátkai ◽

...

Keyword(s):

Ampa Receptor ◽

Neuronal Plasticity ◽

Tyrosine Kinases ◽

Hippocampal Neurons ◽

Critical Role ◽

Surface Receptors ◽

Chemically Induced ◽

Cytoskeleton Remodeling ◽

Knockout Animals ◽

Nonreceptor Tyrosine Kinases

Ras and Rab interactor 1 (RIN1) is predominantly expressed in the nervous system. RIN1-knockout animals have deficits in latent inhibition and fear extinction in the amygdala, suggesting a critical role for RIN1 in preventing the persistence of unpleasant memories. At the molecular level, RIN1 signals through Rab5 GTPases that control endocytosis of cell-surface receptors and Abl nonreceptor tyrosine kinases that participate in actin cytoskeleton remodeling. Here we report that RIN1 controls the plasticity of cultured mouse hippocampal neurons. Our results show that RIN1 affects the morphology of dendritic protrusions and accelerates dendritic filopodial motility through an Abl kinase–dependent pathway. Lack of RIN1 results in enhanced mEPSC amplitudes, indicating an increase in surface AMPA receptor levels compared with wild-type neurons. We further provide evidence that the Rab5 GEF activity of RIN1 regulates surface GluA1 subunit endocytosis. Consequently loss of RIN1 blocks surface AMPA receptor down-regulation evoked by chemically induced long-term depression. Our findings indicate that RIN1 destabilizes synaptic connections and is a key player in postsynaptic AMPA receptor endocytosis, providing multiple ways of negatively regulating memory stabilization during neuronal plasticity.

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ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423201112 ◽

2015 ◽

Vol 112 (15) ◽

pp. 4642-4647 ◽

Cited By ~ 52

Author(s):

Henrik M. Hammarén ◽

Daniela Ungureanu ◽

Jean Grisouard ◽

Radek C. Skoda ◽

Stevan R. Hubbard ◽

...

Keyword(s):

Tyrosine Kinases ◽

Critical Role ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Nucleotide Binding ◽

Inhibitory Effect ◽

Atp Binding ◽

Type I ◽

Dynamic Simulations

Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase–kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.

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